Synthesis of ethyleneimine dimer

ABSTRACT

This invention provides a method for synthesizing ethyleneimine dimer which includes reacting 2-(2-aminoethylamino)ethanol with an aqueous HX solution to produce N-(2-haloethyl)-1,2-ethanediamine dihydrohalide; reacting this product with a base in a solvent to convert the N-(2-haloethyl)-1,2-ethanediamine dihydrohalide into ethyleneimine dimer; and purifying the dimer from the solvent. This method of synthesis provides several advantages over previous methods: (1) The starting compounds are all relatively inexpensive; (2) the yield of the product is greater than 20% of the theoretical yield; and (3) the steps of the synthesis are easy, inexpensive and amenable to large-scale production. All of these advantages allow for less expensive production of ethyleneimine dimer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/117,789, filed Jan. 29, 1999.

BACKGROUND OF THE INVENTION

The invention relates to methods for synthesizing compounds for theselective modification of nucleic acids in biological compositions.

The transmission of viral diseases (e.g., hepatitis A, B, and C,acquired immunodeficiency syndrome, and cytomegalovirus infections) byblood or blood products is a significant problem in medicine. Otherbiological compositions, such as mammalian and hybridoma cell lines,products of cell lines, milk, colostrum, and sperm, can also containinfectious viruses. Screening donor biological compositions for viralmarkers can help reduce the transmission of viruses to recipients, butmany screening methods are directed to only a few discrete viruses, andare therefore incomplete, and may also be less than 100% sensitive. Itis therefore important to inactivate viruses contained in donor blood,blood products, or other biological compositions.

A number of agents that are capable of inactivating viruses in bloodhave been developed. For example, ethyleneimine monomer andethyleneimine oligomers (including dimers, trimers, and tetramers) arevery effective viral inactivating agents. Methods for usingethyleneimine oligomers for inactivating viruses in biologicalcompositions are described in U.S. Ser. No. 09/005,606 (filed Jan. 12,1998), hereby incorporated by reference.

SUMMARY OF THE INVENTION

In general, the invention provides a method for synthesizingethyleneimine dimer (1-aziridineethanamine) which includes reacting2-(2-aminoethylamino)ethanol with an aqueous HX solution, where X is ahalogen, to produce N-(2-haloethyl)-1,2-ethanediamine dihydrohalide,reacting the N-(2-haloethyl)-1,2-ethanediamine dihydrohalide with a basein a solvent to convert the N-(2-haloethyl)-1,2-ethanediaminedihydrohalide into ethyleneimine dimer, and then purifying the dimerfrom the solvent (e.g., by continuous extraction).

In preferred embodiments, the halogen is bromine (most preferred),chlorine, fluorine, or iodine. In another preferred embodiment, the HXis diluted, such that the solution is 30-55% (w/w) HX. In yet anotherpreferred embodiment, the solvent includes a C₁₋₆ alcohol (e.g., ethanolor methanol) or water.

In other preferred embodiments, the HX is added drop-wise to the2-(2-aminoethylamino)ethanol, the temperature of the2-(2-aminoethylamino)ethanol is less than 10° C. during the addition ofthe HX, and the reaction of 2-(2-aminoethyl amino)ethanol with HXincludes the step of refluxing. Preferably, the yield of theN-(2-haloethyl)-1,2-ethanediamine dihydrohalide from this reaction is atleast 50% of the theoretical yield. More preferably, the yield is atleast 75% of the theoretical yield.

In still other preferred embodiments, reacting theN-(2-haloethyl)-1,2-ethanediamine dihydrohalide to produce ethyleneiminedimer includes the steps of refluxing and distillation. Preferably, theyield of ethyleneimine dimer from this reaction is at least 20% of thetheoretical yield. More preferably, the yield is at least 25% of thetheoretical yield, and most preferably, the yield is at least 30% of thetheoretical yield.

Preferably, the ethyleneimine dimer which results from the method ofsynthesis is at least 90% pure, more preferably at least 95% pure, andmost preferably at least 98% pure.

The method of synthesis described herein provides several advantagesover previous methods: (1) The starting compounds are all relativelyinexpensive; (2) the yield of product is greater than 20% of thetheoretical yield; and (3) the steps of synthesis are easy, inexpensiveand amenable to large-scale production. All of these advantages allowfor less expensive production of ethyleneimine dimer.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

DETAILED DESCRIPTION

An example of the synthesis is provided below. From the descriptionprovided herein, one skilled in the art can easily ascertain theessential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

EXAMPLE

Synthesis of Ethyleneimine Dimer

Step 1: Synthesis of N-(2-Bromoethyl)-1,2-Ethanediamine Dihydrobromidefrom 2-(2-Aminoethylamino)Ethanol

N-(2-bromoethyl)-1,2-ethanediamine dihydrobromide is synthesized asfollows. 100 mL of chilled (0.99 moles) of 2-(2-aminoethylamino)ethanolis placed in an ice bath for about 30-45 minutes. IL (8.84 moles) of 48%(w/w) hydrobromic acid is added dropwise over 1.5 hours. This mixture isdistilled in a vigreux distillation apparatus until the temperaturedistillate is 124° C. (three hours), refluxed for four hours, distilledto 124° C. (1.5 hours), refluxed for four hours, distilled to 124° C.(30 minutes), refluxed for four hours, distilled to 124° C. (30minutes), refluxed for four hours, and distilled to 124° C. (10minutes). The remainder of the HBr is removed when the mixture is cooledand concentrated on a rotary evaporator under vacuum at 6 mm pressure.The resulting residue is dissolved in boiling EtOH/H₂O (1175 mL/125 mL)and allowed to crystallize at 4° C. for about 12 hours. The crystals arecollected by filtration, washed with cold ethanol, and thenrecrystallized from EtOH/H₂O as described above. The crystals arecollected by filtration, washed with cold EtOH, and dried in an ovenunder vacuum at 6 mm pressure at 60° C. for 12 hours. The yield isapproximately 254.4 g (78% of theoretical yield) of white solid with amelting point of 170-171° C. This white powder isN-(2-bromoethyl)-1,2-ethanediamine dihydrobromide. Thin layerchromatography shows trace of a more-polar impurity.

A higher percent yield of N-(2-bromoethyl)-1,2-ethanediaminedihydrobromide can be obtained by allowing the pot residue remainingafter the final vigreux distillation to cool to 95° C., and then addingto this pot residue 100 mL of 100% ethanol.N-(2-bromoethyl)-1,2-ethanediamine dihydrobromide will crystallizeovernight. The solid is collected and washed several times with ice cold100% ethanol. This yield is approximately 87% of theoretical yield.

The amount of HBr can be reduced to approximately 4 moles for every 1mole of 2-(2-aminoethylamino)ethanol, thus reducing the cost ofsynthesis.

Step 2: Synthesis of Ethyleneimine Dimer fromN-(2-Bromoethyl)-1,2-Ethanediamine Dihydrobromide

Ethyleneimine dimer is synthesized as follows. Sodium hydroxide (32.04g) is dissolved in 50 mL deionized water and chilled in an ice bath tobelow 25° C. To this solution is added 65.78 g (0.2 moles) ofN-(2-bromoethyl)-1,2-ethanediamine dihydrobromide and 250 mL ethanol.The reaction mixture is refluxed for about one hour. The reactionmixture is cooled, then distilled in a vigreux distillation apparatusunder reduced pressure to remove the bulk of the ethanol. The potresidue is loaded into a continuous extractor and extracted with etherfor 42 hours. The ether extract is dried over sodium sulfate, filtered,and then distilled through a vigreux column first under argon and thenunder reduced pressure. Fractions with a high percentage of dimer (asdetermined by gas chromatography) are further distilled two or moretimes (the final distillation from sodium) to give 5.2 g of a clear,colorless liquid, with a boiling point of 78-80° C. at 138 mm Hg. Thisliquid is 99.3% ethyleneimine dimer by gas chromatography, correspondingto a 30% yield (2.6% water by Karl Fischer water determination).

Ethyleneimine dimer can also be synthesized fromN-(2-bromoethyl)1,2-ethanediamine dihydrobromide as follows. 1.152 kg ofsodium hydroxide (28.8 moles) is placed into a 12 L three-neck roundbottom flask. To this is added 5.85 L of HPLC-grade methanol. Thereaction mixture is cooled for about two hours to 8° C. 2.367 kg (7.2moles) of N-(2-bromoethyl)-1,2-ethanediamine dihydrobromide is thenadded to the reaction mixture over 15 minutes. The temperature should beabout 9° C. at the end of the addition. The reaction mixture is refluxedfor one hour, and then cooled to room temperature. The solid is removedby filtration, and the methanol is distilled from the filtrate underargon atmosphere. The distillate should be about 5.52 L. The pot residueis cooled to room temperature under argon and additional solid isremoved by filtration. The filtrate is cooled in a 4° C. refrigeratorfor four hours, and newly formed solid is removed by filtration. Thefiltrate is distilled through a vigreux column under vacuum (138 mm Hg)and fractions are collected. Ethyleneimine content is determined usinggas chromatography. Fractions with a high percentage of dimer (asdetermined by gas chromatography) are distilled to purity, as describedabove, resulting in 186 g of a clear, colorless liquid with a boilingpoint of 78-80° C. at 138 mm Hg. The yield is 30% of theoretical yield.

The purity of the synthesized ethyleneimine dimer is determined usingstandard methods of gas chromatography known to those skilled in theart. A suitable column is a RESTEK RTX®-5.15 m×0.53 mm×1.0 μm analyticalcolumn, compatible with, for example, a Hewlett Packard Model 6890Series with FID detection. Using the method of synthesis describedherein, the ethyleneimine dimer is at least 98% pure. Thin layerchromatography shows piperazine as an impurity.

What is claimed is:
 1. A method of synthesizing ethyleneimine dimer,said method comprising the steps: (a) reacting2-(2-aminoethylamino)ethanol with an aqueous HX solution, where X is ahalogen, to produce N-(2-haloethyl)-1,2-ethanediamine dihydrohalide; (b)reacting said N-(2-haloethyl)-1,2-ethanediamine dihydrohalide with abase in a solvent to convert said N-(2-haloethyl)-1,2-ethanediaminedihydrohalide into ethyleneimine dimer; and (c) purifying saidethyleneimine dimer from said solvent.
 2. The method of claim 1, whereinX is Br, Cl, I, or F.
 3. The method of claim 2, wherein X is Br.
 4. Themethod of claim 1, wherein said HX solution is 30-55% (w/w) HX.
 5. Themethod of claim 1, wherein said solvent comprises water or a C₁₋₆alcohol.
 6. The method of claim 1, wherein said HX solution is added tosaid 2-(2-aminoethylamino)ethanol such that the temperature of thereaction mixture is less than 10° C. during said addition.
 7. The methodof claim 1, wherein said HX is added drop-wise to said2-(2-aminoethylamino)ethanol.
 8. The method of claim 1, wherein saidreacting 2-(2-aminoethyl amino)ethanol with HX comprises the step ofrefluxing.
 9. The method of claim 1, wherein the yield of saidN-(2-haloethyl)-1,2-ethanediamine dihydrohalide from said2-(2-aminoethylamino)ethanol is at least 50% of the theoretical yield.10. The method of claim 1, wherein the yield of saidN-(2-haloethyl)1,2-ethanediamine dihydrohalide from said2-(2-aminoethylamino)ethanol is at least 75% of the theoretical yield.11. The method of claim 5, wherein said reacting saidN-(2-haloethyl)-1,2-ethanediamine dihydrohalide in said C₁₋₆ alcohol toproduce ethyleneimine dimer comprises the step of refluxing.
 12. Themethod of claim 1, wherein said reacting saidN-(2-haloethyl)-1,2-ethanediamine dihydrohalide to produce ethyleneiminedimer comprises the step of distillation.
 13. The method of claim 1,wherein said purifying comprises a continuous extraction.
 14. The methodof claim 1, wherein said purifying comprises a series of distillationsand filtrations.
 15. The method of claim 1, wherein said ethyleneiminedimer is at least 90% pure after said purifying step (c).
 16. The methodof claim 1, wherein said ethyleneimine dimer is at least 95% pure aftersaid purifying step (c).
 17. The method of claim 1, wherein saidethyleneimine dimer is at least 98% pure after said purifying step (c).18. The method of claim 1, wherein the yield of said ethyleneimine dimerfrom said N-(2-haloethyl)-1,2-ethanediamine dihydrohalide is at least20% of the theoretical yield.
 19. The method of claim 1, wherein theyield of said ethyleneimine dimer from saidN-(2-haloethyl)-1,2-ethanediamine dihydrohalide is at least 25% of thetheoretical yield.
 20. The method of claim 1, wherein the yield of saidethyleneimine dimer from said N-(2-haloethyl)-1,2-ethanediaminedihydrohalide is at least 30% of the theoretical yield.
 21. A method ofsynthesizing ethyl ethyleneimine dimer, said method comprising thesteps: (a) reacting 2-(2-aminoethylamino)ethanol with HBr to produceN-(2-bromoethyl)-1,2-ethanediamine dihydrobromide; and (b) reacting saidN-(2-bromoethyl)-1,2-ethanediamine dihydrobromide with a base in asolvent having a pH>7.5 to convert saidN-(2-bromoethyl)-1,2-ethanediamine dihydrobromide into ethyleneiminedimer.